Automated surface acquisition for a confocal microscope

Abstract

A method is described for obtaining an image of a target surface with a confocal microscope. The surface to be imaged is represented by a number of points on the surface, each of which has a unique location represented by X, Y, and Z Cartesian coordinates. The microscope selects a starting position for an objective lens of the microscope along a Z vector substantially normal to the surface. The objective lens has a preselected range of travel along the Z vector that is divided into a number of Z positions. Next, the objective lens is positioned and the surface scanned at each of the Z positions. The scan at each Z position provides signals, one for each point on the surface, representing the reflected intensity of laser light. Then, for each point on the surface, the microscope finds the Z coordinate of the point by determining which Z position resulted in the greatest return intensity of reflected laser light. From this information the Z coordinate of any particular point may be determined because the maximum reflected intensity for a given point, when correlated to the Z position of the objective lens, gives the Z location of that point on the surface. Having determined the Z locations for each point on the surface, the Z locations are compared to determine the low and high points on the surface. A second scan is then set up using the low and high points on the surface to determine the optimal scan parameters.

What is claimed is: 
     
       1. A method of obtaining an image of a surface with a confocal microscope having an objective lens, the surface comprised of a plurality of points, each of the points having a unique location represented by X, Y, and Z Cartesian coordinates, the method comprising the steps of: selecting a first starting position for the objective lens along a Z vector from which to scan the surface, the Z vector substantially normal to the surface, the objective lens having a range of travel relative to the surface along the Z vector beginning at the first starting position;   dividing the range of travel into a plurality of Z positions along the Z vector, the plurality of Z positions including the starting position;   for each of the plurality of Z positions, positioning the objective lens at the Z position and scanning the surface to generate a plurality of signals, each of the signals corresponding to a given one of the points on the surface and representing an intensity of light reflected through the objective lens from the given point;   for each of the points on the surface, finding the Z coordinate of the point by determining which of the plurality of Z positions results in a maximum signal for the point, wherein the maximum signal, when correlated to the Z position of the objective lens, gives the Z location of the point on the surface;   comparing the Z locations of the points on the surface to determine a low point on the surface and a high point on the surface; and   selecting a second starting position and a stopping position based on the high point and the low point.   
     
     
       2. The method of claim 1, wherein the microscope includes a photodetector having variable gain, the method further comprising the step of setting the photodetector gain between predetermined limits before scanning the surface. 
     
     
       3. The method of claim 1, wherein the step of selecting a first starting position includes the step of automatically focusing the microscope on the surface. 
     
     
       4. The method of claim 2, wherein each of the maximum signals represents a maximum intensity value of reflected light for a particular point on the surface, the method further comprising the step of smoothing the maximum intensity values by dividing the maximum intensity values into groups of maximum intensity values and averaging the maximum intensity values in each group to create a collection of group intensity values. 
     
     
       5. The method of claim 4, further comprising the steps of: comparing the group intensity values to determine a maximum group intensity value; and   resetting the photodetector gain based on the maximum group intensity value.   
     
     
       6. The method of claim 4, further comprising the steps of: comparing each of the group intensity values to a threshold intensity value; and   for each group intensity value below the threshold intensity value, setting each of the Z values corresponding to maximum intensity values in the group to an average Z value.   
     
     
       7. The method of claim 1, the step of selecting a second starting position and a second scan range further comprising the steps of: calculating a safety margin using the high point and the low point;   adding the safety margin to the high point to determine the second starting point; and   subtracting the safety margin from the low point to determine the stopping point.   
     
     
       8. The method of claim 7, wherein the safety margin is 2.5 microns plus twenty percent (20%) of the difference between the high point and the low point. 
     
     
       9. The method of claim 1, wherein the Z locations of the points on the surface are stored as an array of Z values. 
     
     
       10. The method of claim 9 further comprising the step of smoothing the Z values by dividing the array of Z values into groups of Z values and averaging the Z values in each of the groups to create a collection of group-averaged Z values.